Heterologous expression of Na+-K+-ATPase in insect cells:: intracellular distribution of pump subunits
Heterologous expression of Na+-K+-ATPase in insect cells:: intracellular distribution of pump subunits
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DOI：
10.1152/ajpcell.2001.281.3.c982
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发表时间：
2001-09-01
影响因子：
5.5
通讯作者：
Kaplan, JH
中科院分区：
文献类型：
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作者：
Gatto, C;McLoud, SM;Kaplan, JH
Na+-K+-ATP酶是一种异源二聚体质膜蛋白，在几乎所有动物细胞中负责细胞离子稳态。已经表明，一些昆虫细胞（例如，High Five细胞）没有（或极低）Na+-K+-ATP酶活性。我们通过杆状病毒表达系统在High Five细胞中表达了羊肾Na+-K+-ATP酶α和β亚基。我们使用定量狭缝印迹分析，以确定表达的Na+-K+-ATP酶包括在这些细胞中的总膜蛋白的0.5%和2%之间。使用五步蔗糖梯度（0.8-2.0 M）分离的内质网，高尔基体，和质膜组分，我们观察到功能Na+泵分子在每个膜池和其特性。几乎所有表达的蛋白质功能正常，类似于在纯化的狗肾酶制剂中发现的。因此，这里描述的测量并不复杂的非功能性异源表达的酶的丰度。具体而言，哇巴因敏感的ATP酶活性，[H-3]哇巴因结合，和阳离子依赖性进行了测量，为每个馏分。Na+-K+-ATP酶的功能特性在内质网中组装后基本不变。此外，我们测量了整个细胞中哇巴因敏感的Rb-86（+）摄取，作为特异性评估正确折叠并递送至质膜的Na+-K+-ATP酶分子的手段。当α亚基或β亚基单独表达时，我们不能测量任何哇巴因敏感活性。分离的膜级分的免疫染色表明，当单独表达时，α-亚基在蛋白质成熟途径的早期被降解（即，内质网），但β-亚基被正常加工并递送至质膜。因此，似乎只有α-亚单位具有成熟和运输到质膜的寡聚体要求。此外，α-β异二聚体在内质网内的组装显然不需要Na+泵特异性伴侣。
The Na+-K+-ATPase is a heterodimeric plasma membrane protein responsible for cellular ionic homeostasis in nearly all animal cells. It has been shown that some insect cells (e.g., High Five cells) have no (or extremely low) Na+-K+-ATPase activity. We expressed sheep kidney Na+-K+-ATPase alpha- and beta -subunits individually and together in High Five cells via the baculovirus expression system. We used quantitative slot-blot analyses to determine that the expressed Na+-K+-ATPase comprises between 0.5% and 2% of the total membrane protein in these cells. Using a five-step sucrose gradient (0.8-2.0 M) to separate the endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions, we observed functional Na+ pump molecules in each membrane pool and characterized their properties. Nearly all of the expressed protein functions normally, similar to that found in purified dog kidney enzyme preparations. Consequently, the measurements described here were not complicated by an abundance of nonfunctional heterologously expressed enzyme. Specifically, ouabain-sensitive ATPase activity, [H-3]ouabain binding, and cation dependencies were measured for each fraction. The functional properties of the Na+-K+-ATPase were essentially unaltered after assembly in the endoplasmic reticulum. In addition, we measured ouabain-sensitive Rb-86(+) uptake in whole cells as a mean to specifically evaluate Na+-K+-ATPase molecules that were properly folded and delivered to the plasma membrane. We could not measure any ouabain-sensitive activities when either the alpha -subunit or beta -subunit were expressed individually. Immunostaining of the separate membrane fractions indicates that the alpha -subunit, when expressed alone, is degraded early in the protein maturation pathway (i.e., the endoplasmic reticulum) but that the beta -subunit is processed normally and delivered to the plasma membrane. Thus it appears that only the alpha -subunit has an oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the alpha-beta heterodimer within the endoplasmic reticulum apparently does not require a Na+ pump-specific chaperone.